Comparative study of the effect of different extracts of M ...used to evaluate the significance of...

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Journal of Medicinal Plants Studies 2018; 6(6): 84-90

ISSN (E): 2320-3862

ISSN (P): 2394-0530

NAAS Rating: 3.53

JMPS 2018; 6(6): 84-90

© 2018 JMPS

Received: 14-09-2018

Accepted: 18-10-2018

Abubakar Amali Muhammad

Department of Pharmacology,

Faculty of Pharmaceutical

Sciences, Usmanu Danfodiyo

University Sokoto, Nigeria

Dallatu Muhammad Kabir

Department of Chemical

Pathology, Faculty of Medical

Laboratory Science, Usmanu

Danfodiyo University Sokoto,

Nigeria

Asmau Darma Lawal

Department of Chemical

Pathology, Faculty of Medical

Laboratory Science, Usmanu

Danfodiyo University Sokoto,

Nigeria

Correspondence

Abubakar Amali Muhammad

Department of Pharmacology,

Faculty of Pharmaceutical

Sciences, Usmanu Danfodiyo

University Sokoto, Nigeria

Comparative study of the effect of different

extracts of M. Oleifera on some biochemical and

histological parameters in rats

Abubakar Amali Muhammad, Dallatu Muhammad Kabir and Asmau

Darma Lawal

Abstract Moringa oleifera is a plant whose therapeutic significance has been reported scientifically. However,

relatively few studies elucidate the safety of different extraction solvents used for the extraction of the

plant bio actives. This study investigated the effect of administration of different solvent extracts of M.

oleifera on liver and kidney functions of rats. Thirty six adult male rats were used and divided into four

groups of nine rats each. Group 1 served as control. Different solvent extracts (aqueous, ethanol, and

methanol) were administered to study groups (2, 3 and 4) at different doses of 100, 200 and 400 mg/kg

respectively. Biochemical parameters such as total protein, albumin, total and conjugated bilirubin, AST,

ALT, urea and creatinine were determined after 3 days (acute) and 28 days (sub-chronic) using standard

techniques. Histology of liver and kidney tissues was examined 28 days after administration of the

extracts at the highest dose of 400mg/kg. The results showed treatment-related abnormalities in most of

the biochemical parameters of methanolic and ethanolic extracts of M. oleifera. However, aqueous

extracts did not show any abnormality on the biochemical parameters. Methanolic and ethanolic extracts

of M. oleifera significantly increased (p<0.05) the activity of aspartate amino transferase, alanine

aminotransferase, total bilirubin, urea and creatinine. It also showed significant decrease (p<0.05) in the

levels of total protein and albumin. The significant increase and decreases were however, dose

dependent. This was supported by the result of histological evaluation of liver and kidney which did not

show any abnormal feature following administration of aqueous extract of M. oleifera. The present study

demonstrated some level of safety in the use of aqueous extract of M. oleifera when compared to

methanolic and ethanolic extracts that revealed some form of abnormality in the kidney and liver tissues

following 28 days of administration. This suggests that, care should be taken in the use of these extracts

for their counterproductive consequences resulting possibly from chronic administration of the extract.

Keywords: comparative, M. Oleifera, histological parameters, rats

Introduction

Moringa oleifera (Moringaceae) is a valuable plant, distributed in many tropical and

subtropical countries. It has variety of medicinal uses. Different parts of this plant contain

minerals, proteins, vitamins, B – carotene, amino acids and various phenolics (Bukar et al.,

2010) [3].

Pharmacologically, M. oleifera has been reported to be useful in the treatment and prevention

of disease or infection either by using dietary or tropical administration of Moringa

preparations such as (Extracts, decoction, poultices, creams, oils, emollients, salves, powders

and porridges) (Palada, 1996) [14]. Moringa oleifera leaves has been reported to possess

antimicrobial action (Prashith et al., 2010) [16] anti-inflammatory properties (Mahajan, et

al.,2007) [11], antidiabetic (Jaiswal et al., 2009; Edoga, et al., 2013) [9, 7], antioxidant (Sultana et

al., 2009) [19], and anticancer properties (Parvathy and Umamaheshwari, 2007; Sreelatha, et al.,

2011) [15, 18] and recently, it has been reported to possess wound healing properties both in vitro

and in vivo.

However, this study was undertaken to compare the toxicological effects of different

extraction solvent of Moringa oleifera leaves on histological appearance of the vital organs

and some biochemical parameters.

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Journal of Medicinal Plants Studies

Materials and Methods

Collection of Plant material

Fresh leaves of M. oleifera were purchased from Kara Market

in Sokoto town of Sokoto State, Nigeria. The leaves were air

dried for about one week away from direct sunlight to avoid

possible damage to their phyto-constituents. The leaves were

grinded in to fine powder and constant weight was obtained

and stored appropriately until needed for extraction.

Extraction of Plant material

The extraction was carried out by maceration process in

which 500g each of the dried leaves powder were soaked in

1000 ml of 70% ethanol. 80% methanol and distilled water

using separate 5 litres volumetric flask. The mixture was

shaken for 30minutes and then allowed to stay at room

temperature overnight. The filtrate was decanted in a separate

container. This process was repeated for 3 consecutive days to

ensure complete extraction. The mixtures were first filtered

with cheese cloth, then with What Man No 1 filter paper. The

filtrates were separately concentrated in vacuum using Rotary

Evaporator (Model EYELA SB- 1100, China) to 10% of its

original volume at 60oC. This was concentrated to complete

dryness in water bath at 45°C in order to obtain the crude

extract and the dried extact was stored at 4°C until needed for

further use.

Grouping and treatment of animals

Thirty adult male Wistar rats weighing (200-300g) were

obtained from the animal house of the Faculty of

Pharmaceutical Sciences, Usman Danfodiyo University

Sokoto. Nigeria and were allowed to acclimatize for a period

of fourteen days in well ventilated room with a temperature

and relative humidity of 29±2ºc. They were maintained with

commercial rat chow (Vital Feeds LMT, Jos, Plateau,

Nigeria) and water and libitum. The animals were housed in a

cage and were exposed to 12 hour light-dark cycle and

handled according to NIH standard protocol. At the end of the

acclimatization period, they were divided into four groups of

nine rats per group. Group 1 served as control and were

treated with distilled water of treatment equivalence, group 2,

3 and 4, were treated with aqueous, ethanol, and methanol of

the crude extract of M. oleifera respectively at different doses.

The administration of the extract lasted for twenty eight days

period.

Group 2, were administered with aqueous extract of M.

oleifera leaves at different dose of 100 mg/kg, 200 mg/kg and

400 mg/kg. Group 3 were administered with ethanolic extract

of M. oleifera leaves at different doses of 100mg/kg,

200mg/kg and 400mg/kg. Group 4 were also treated with

methanolic extract of M. oleifera leaves at the same dosage

range as above intra gastric gavage using oral cannula (a

feeding needle).

Collection of blood

Three days after administration of the extracts, the animals

were anaesthetized using chloroform, and the blood samples

were collected from the animals through cardiac puncture,

into clean, dry plain containers. The serum of each sample

was analysed for biochemical parameters. The procedure was

repeated after 28 days administration of the extracts. The

blood collected was allowed to clot and then centrifuged at

5000 rpm for 10 minutes. The serum of each sample was used

for the assay of aspartate amino transferase (AST), alanine

anpmino transferase (ALT), total protein (TP), albumin

(ALB), globulin (GLOB), urea and creatinine (Cr).

Histopathological Examinations

The Liver and Kidney of the rats were fixed and processed

carefully Sections of 5-6μm in thickness were cut and made

onto slides. These tissue sections were then stained using

Haematoxyline and Eosin method for photo-microscopic

examination of general tissue structures as reported by Orchad

and Nation (2012) [10].

Statistical analysis

The data obtained from this study was analyzed using the

statistical package for social science (SPSS) for Windows,

version 21.0 (SPSS Inc., Chicago, IL, USA). The data was

represented as mean ± standard deviation (S.D). ANOVA was

used to evaluate the significance of the difference between the

mean values of the measured parameters in the respective test

and control groups. A mean difference was considered

significant when p < 0.05.

Results

The results of biochemical analysis of the liver enzymes

showing a slight increase in serum levels of alanine amino

transferase (ALT) and aspartate amino transferase (AST)

following 3 days administration of different solvent extracts

of M. oleifera at different doses of 100mg/kg, 200mg/kg and

400mg/kg as compared with the control. (Figure 4.1).

Fig 4.1: Effect of different extracts of M. oleifera on AST and ALT,

following 3 days administration at different doses. **Significantly

different from control (P<0.05)

The results of biochemical analysis of the liver enzymes

showing a significant increase in serum levels of alanine

amino transferase (ALT) and aspartate amino transferase

(AST) following 28 days administration of different solvent

extracts of M. oleifera at different doses of 100mg/kg,

200mg/kg and 400mg/kg as compared with the control.

(Figure 4.2).

Fig 4.2: Effect of different extracts of M. oleifera on AST and ALT

following 28 days after administration at different doses.

**Significantly different from control (P<0.05)

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The result of the biochemical analysis showing a slight

increase in serum levels of total bilirubin (TB) and direct

bilirubin (DB) following 3 days of administration of aqueous


200mg/kg and 400mg/kg as compared with their controls

(Figure 4.3).

Fig 4.3: Effect of different extracts of M. oleifera on total and direct bilirubin 3 days after administration at different doses.



increase in serum levels of total bilirubin (TB) and direct

bilirubin (DB) following 28 days of administration of aqueous



(Figure 4.4)

Fig 4.4: Effect of different extracts of M. oleifera on total and direct bilirubin 28 days after administration of different doses. **Significantly



increase in serum levels of total protein, Albumin and

Globulin) following 3 days administration of aqueous extracts

of M. oleifera at different doses of 100mg/kg, 200mg/kg and

400mg/kg as compared with their controls (Figure 4.5).

Fig 4.5: Effect of different extracts of M. oleifera on total protein, Albumin and Globulin 3 days following administration at different doses.

**Significantly different from extract administered groups (P< 0.05)

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increase in serum levels of total protein, Albumin and

Globulin) following 28 days administration of aqueous



(Figure 4.6).

Fig 4.6: Showing the effect of different solvent extract of M. oleifera

on total protein, Albumin and Globulin 28 days after administration

at different doses. **Significantly different from extract

administered groups (P<0.05)


increase in serum levels of Urea and Creatinine following 3

days administration of aqueous extracts of M. oleifera at

different doses of 100mg/kg, 200mg/kg and 400mg/kg as

compared with their controls (Figure 4.7).

Fig 4.7: Effect of different solvent extract of M. oleifera on Urea and

Creatinine 3 days after administration at different doses.



increase in serum levels of Urea and Creatinine following 28

days administration of aqueous extracts of M. oleifera at

different doses of 100mg/kg, 200mg/kg and 400mg/kg as

compared with their controls (Figure 4.8).

Fig 4.8: Effect of different extracts of M. oleifera on Urea and Creatinine 28 days after administration at different doses. **Significantly


The result of histopathological examinations of the liver

sections under the light microscope revealed normal

hepatocellular tissue, in normal control group. (Figure 4.9).

Fig 4.9: Group 1 (Control) Showing normal liver cells (M x 640, H

and E)

The result of histopathological examinations of the Kidney

sections under the light microscope revealed normal kidney

tissue, in normal control group. (Figure 4.9).

Fig 4.10: Group 1 (Control) Showing normal kidney tissue (M x

640, H and E)


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sections under the light microscope revealed normal

hepatocellular tissue, 28 days after administration of aqueous

extract of M. oleifera at 400mg/kg body weight/ day as

compared with the control group. (Figure 4.11).

Fig 4.11: showing normal hepatocellular tissue, 28 days after

administration of aqueous extract of M. oleifera at 400mg/kg (M x

640, H and E)


sections under the light microscope revealed normal kidney

tissue, 28 days after administration of aqueous extract of M.

oleifera at 400mg/kg body weight/ day as compared with the

control group. (Figure 4.12).

Fig 4.12: showing normal kidney tissue, 28 days after administration

of aqueous extract of M. oleifera at 400mg/kg (M x 640, H and E)


sections under the light microscope revealed cytoplasmic

degeneration of hepatic cells, 28 days after administration of

aqueous extract of M. oleifera at 400mg/kg body weight/ day

as compared with the control group. (Figure 4.13).

Fig 4.13: showing preserved hepatocellular architecture with

cytoplasmic degeneration of hepatic cells hypochromic 28 days after

administration of ethanolic extract of M. oleifera at 400mg/kg (M x

640, H and E)


sections under the light microscope revealed kidney tissue

with glomerular inflammation 28 days after administration of

aqueous extract of M. oleifera at 400mg/kg body weight/ day

as compared with the control group. (Figure 4.14).

Fig 4.14: showing kidney tissue with glomerular inflammation 28

days after administration of ethanolic extract of M. oleifera at

400mg/kg (M x 640, H and E)


sections under the light microscope revealed hepatocellular

tissue with evidence of intrahepatic hemorrhage following 28

days administration of aqueous extract of M. oleifera at

400mg/kg body weight/ day as compared with the control

group. (Figure 4.15)

Fig 4.15: showing hepatocellular tissue with evidence of intrahepatic

hemorrhage 28 days after administration of ethanolic extract of M.

oleifera at 400mg/kg (M x 640, H and E)


sections under the light microscope revealed mild glomerular

congestion 28 days after administration of aqueous extract of

M. oleifera at 400mg/kg body weight/ day as compared with

the control group. (Figure 4.16).

Fig 4.16: showing mild glomerular congestion 28 days after

administration of methanolic extract of M. oleifera at 400mg/kg (M

x 640, H and E)

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Discussion

The result of the present study revealed a slight increase in

serum levels of alanine amino transferase (ALT), aspartate

amino transferase (AST), Total bilirubin (TB) and direct

bilirubin (DB) after 3 and 28 days of administration of

aqueous extracts of M. oleifera at different doses of

100mg/kg, 200mg/kg and 400mg/kg as compared with their

controls. This result is in contrast with the study of Adedapo

et al., 2009 in which they showed significant increase in the

activity of liver enzymes AST, ALT when ethanolic and

aqueous leaves extract of M. oleifera was administered to rats

at the dose of 1.6g/kg. This contrast may be due to variation

in the dosage of the extracts administered.

However, following 3 days and 28 days administration of

aqueous extract of M. oleifera, there was no significant

increase or decrease (p>0.05) in total protein, albumin,

globulins, urea and creatinine at the doses of 100mg/kg,

200mg/kg and 400mg/kg when compared with the control this

signifies that, aqueous extract of M. oleifera leaves did not

interfere with the synthetic functions of the liver. The slight

decrease in serum urea and creatinine signifies that it is non-

toxic to the kidney as reported earlier by Abdulazeez et al.,

2010. This finding from biochemical analysis also supports

the result of histology conducted on the liver and kidney

which showed no abnormal feature in these organs following

administration of aqueous extract of M. oleifera at the dose of

400mg/kg body weights of rats.

Administrations of ethanolic extract of M. oleifera

significantly caused increase (p<0.05) in serum levels of

ALT, AST and Total Bilirubin after 3 and 28 days at the

doses of 100mg/kg, 200mg/kg and 400mg/kg as compared

with their controls. These increases may be as a result of

cellular leakage and loss of functionality of membrane

integrity as reported by Saraswat et al., 1993. These results

does not agree with the study of Nwangwu et al., 2010 which

showed significant reductions in ALT, AST, ALB and T.BIL

and increase in TP when 200mg/kg ethanolic extract

Gongronema latifolium leaves were administered to male rats

However, ethanolic extract of M. oleifera also shows a

significant reduction (p<0.05) in Total protein, albumin and

globulin both after 3days and 28day of administration at

different doses. The decrease in serum total protein (TP)

observed with the ethanolic extract in this study could be as a

result of reduced albumin (ALB) which is a marker for

diagnosis of liver disease as reported by Benoit et al., 2000.

This decrease could have resulted from a concomitant

decrease in the number of cells responsible for ALB synthesis

in the liver which is in agreement with the report of Omotuyi

et al., 2008 or a direct interference with the albumin-

synthesizing mechanism in the liver as obtainable in

mammalian cells or a combination of both.

Creatinine and urea are major catabolic products of creatin

and protein metabolism, respectively. Increases in serum urea

levels from 28days of administration of the extract can be the

effect of nephrotoxicity of M. oleifera ethanolic leave extract

indicative of impaired kidney function. Afolayan and Yakubu,

2009; Abdulazeez et al., 2010 stated that renal failure leads to

retention of creatinine and other non-protein nitrogenous

constituents of the blood which may be responsible for the

increases observed with the ethanolic extract groups in this

study.

These increases in serum enzymes level, Creatinine and urea

and decrease in the level of protein may be accounted for the

changes found in the liver and kidney cells of ethanolic

extract of M. oleifera at 400mg/kg concentration.

Methanolic extract of M. Oleifera significantly increased

(p<0.05) serum levels ALT, AST and Total Bilirubin after 3

and 28 days of administration of the extracts at the doses of

100mg/kg, 200mg/kg and 400mg/kg as compared with their

controls. This is in agreement with the findings of (Abu et al.,

2013) [2] which showed a significant increase in serum levels

ALT, AST and Total Bilirubin when 300mg/kg of methanolic

extract of H. Polyrhizus fruits was administered to rats. This

could be due to leakage of enzyme from the damage

hepatocytes into systemic circulation.

A significant reduction (p<0.05) in Total protein, albumin and

globulin both after 3days and 28day of administration at

different doses as above was also seen in methanol extract

which could be due to damage of the hepatocytes. The result

also revealed significantly increased in serum urea and

ceratinine following 3 and 28 days of methanolic extract at

different doses when compared with their controls. This

happens when kidneys removal cannot match up with the rate

of production of this product due to kidney disease, high

concentrations are measured. Other factors such as high

protein intake, increased protein catabolism, stress and

dehydration can be responsible for such increases. This may

also be associated with the abnormal features seen in

histology of liver and kidney when 400mg/kg of methanolic

extract of M. oleifera was administered.

Histological analysis of aqueous extract of M. oleifera

showed no abnormal features in the liver and kidney tissues

even at higher dose of 400mg/kg for 28 days. This finding is

in accordance with the study of (Adedapo et al., 2009) [4] who

observed no abnormal features in histopathology of liver and

kidney of rats treated with the dose of 16.1g/kg of aqueous

extract of M. oleifera.

The ethanolic extract of M. oleifera treated rats showed

preserved hepatocellular architecture with cytoplasmic

degeneration of hepatic cell, hypochromic at the dose of

400mg/ kg after 28 days of administration. However, the

kidney tissue showed mild glomerular inflammation at the

dose of 400mg/kg after 28days. This is in agreement with the

findings of (Josephine et al., 2012) [10]. Which showed

congestion with scattered focal necrosis of the liver tissue and

kidney tissue with expanded and congested glomeruli after

receiving a single oral daily dose of ethanol extract of M.

oleifera for 30 days.

The methanolic extract of M. Oleifera showed normal

hepatocellular cells with evidence of intrahepatic

haemorrhage. The kidney tissue also showed mild glomerular

congestion at the dose of 400mg/kg for 28days. This is in

agreement with the findings of Ajibade et al., 2011.Which

showed mild portal cellular infiltration in the liver cell and

kidney tissues showed mild cortical congestion at 400mg/kg

of methanol seed extract of M. oleifera for 30days.

Conclusion

The ethanolic and methanolic extracts of M. oleifera

demonstrated nephrotoxic and hepatotoxic potential which

was not the case with the aqueous extract. This suggests that,

care should be taken in the use of the ethanolic and

methanolic extracts of M. oleifera for the reasons of its

counterproductive consequences resulting possibly from

bioaccumulation as a consequence of prolonged

administration

References

1. Abdulazeez AM, Awasum CA, Dogo YS, Abiayi PN.

Effect of Peristrophe bicalyculata on blood pressure,

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kidney and liver functions of two kidney one clip (2K1C)

hypertensive rats. Brazil Journal Pharmacology and

Toxicology. 2010; 1:101-107.

2. Abubakar AM, Nur AS, Palanisamy A, Faridah A,

Sharida F. In vitro wound healing potential and

identification of bio active compounds from M. oleifere

lam, 2013.

3. Bukar A, Uba A, Oyeyi T. Antimicrobial profile of

Moringa oleifera Lam. extracts against some food–borne

microorganisms. Bayero Journal of Pure and Applied

Sciences. 2010; 3(1).

4. Adedapo AA. Mogbojuri OM, Emikpe BO. Safety

evaluations of the aqueous extract of the leaves of

Moringa oleifera in rats. Journal of Medicinal Plants

Research. 2009; 3:586-591.

5. Afolayan AJ, Yakubu MT. Effect of Bulbine natalensis

baker stem extract on the functional indices and histology

of the liver and kidney of male Wistar rats. Journal of

Medicinal Food. 2009; 12:814-820.

6. Ajibide TO, Olayemi FO, Arowolo RO. The

haematological and biochemical effects of methanol

extract of M. oleifera in rats. Journal of medicinal plant

research. 2012; 6(4):615-621.

7. Edoga C, Njoku O, Amadi E, Okeke J. Blood sugar

lowering effect of Moringa oleifera lam in albino rats,

International Journal of Science and Technology. 2013;

3:1.

8. European Treaty Series. European Convention for the

Protection of Vertebrate animals and other scientific

purposes, 2005, 123.

9. Jaiswal D, Kumar P, Rai A, Kumar S, Watal G. Effect of

Moringa oleifera Lam. leaves aqueous extract therapy on

hyperglycemic rats, Journal of Ethnopharmacology.

2009; 123:392-396.

10. Josephine N, kasolo Gabriel S, Bimeya Lonzi O, Jasper

W. Subacute toxicityevaluation of M. oleifera leaves

aqueous and ethanolic extacts in swiss albino rats.

International journal of medicinal plant research. 2012;

6:75-81

11. Mahajan SG, Mali RG, Mehta AA. Protective effect of

ethanolic extract of seeds of Moringa oleifera lam.

Against inflammation associated with development of

arthritis in rats, Journal of Immunotoxicology. 2005;

4:39-47.

12. Nwangwu SCO, Nwangwu UC, Josiah SJ, Ezenduka C.

Hepatoprotective and hypolipidemic potentials of

aqueous and ethanolic leaf extracts of Gongronema

latifolium on normal male rats. Journal of Science

Engineering Technology. 2010; 17:9572-9583.

13. Omotuyi IO, Nwangwu SC, Okugbo OT, Okoye OT,

Ojieh GC, Wogu DM. Hepatotoxic and hemolytic effects

of acute exposure of rats to artesunate overdose. African

Journal of Biochemistry Research. 2008; 2:107-110.

14. Palada MC. Hort Science 1996; 31:794-797.

15. Parvathy M, Umamaheshwari A. Cytotoxic effect of

Moringa oleifera leaf extracts on human multiple

myeloma cell lines, Trends in Medical Research, 2007,

44-50.

16. Prashith TR, Kekuda N, Mallikarjun D, Swathi KV,

Nayana MB, Rohini TR. Antibacterial and antifungal

efficacy of steam distillate of Moringa oleifera lam,

Journal of Pharmaceutical Sciences and Research. 2010;

2:34-37.

17. Saraswat B, Visen PK, Patnaik GK, Dhawan BN.

Anticholestic effect of picroliv, active hepatoprotective

principle of Picrorhiza kurrooa, against carbon

tetrachloride induced cholestasis. Indian Journal of

Experimental Biology. 1993; 31:316-318.

18. Sreelatha S, Jeyachitra A, Padma PR. Antiproliferation

and induction of apoptosis by Moringa oleifera leaf

extract on human cancer cells, Food and Chemical

Toxicology. 2011; 49:1270-1275.

19. Sultana B, Anwar F, Ashraf M. Effect of extraction

solvent/technique on the antioxidant activity of selected

medicinal plant extracts, Molecules, 2009, 2167-2180.

20. The Wealth of India (A Dictionary of Indian Raw

Materials and Industrial Products). Raw Materials, 1962.

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